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Base Excision Repair
- Involves excision of single or multiple nucleotides
- Followed by re-synthesis of correct nucleotides
- Protects against transitions, Transversions, alkylation, deamination, etc.
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Components of Base Excision Repair (BER)
- 1. DNA glycosylase
- 2. AP endonuclease
- 3. DNA polymerase B
- 4. DNA ligase
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Base Excision Repair mechanism
- Damage detected by glycosylase: cleaves glycosidic bond b/w sugar of nucleotide and DAMAGED base
- AP: site of DNA lacking Base, UNSTABLE and degrades easily, yields GAP in DNA
- AP endonuclease, DNA pol B and ligase now interact to repair
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Uracil DNA glycosylase (UDG) and pinch-push mechanism
- UDG compresses DNA, through phosphate interactions with uracils
- This causes DESTABILIZATION of the base…promoting FLIPPING of uracil out of the DNA base site
- Residue Leu272 of UDG may be involved in PUSHING uracil out (debated)
- Leu272 STABILIZES AP site until DNA Pol can replace base
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Mismatch Repair Role
- to Correct Base Substitution Mismatches and insertion-deletion mismatches generated during replication
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Recognition of mismatches
- Mismatched DNA DESTABILIZES DNA duplex, which kink the DNA at the mismatch site
- Involves MutS, and its Aromatic ring stack with Phe39 and H-bond with Glu41
- Bind to mismatch via minor groove and recognize mismatch thru conserved Phe-X-Glu motif
- MutL (and MutH) act to find mismatch and help cut it out
- Once cut, HELICASE moves the mismatched DNA
- DNAP III inserts correct base
- Ligase connects it
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Example of mismatched recognition with thymine
- Phenylalanine is IMPORTANT in DNA binding and MMR activity!!
- Phe 39 of MutS approaches DNA minor groove and stacks on the unpaired T: resulting in a sharp kink
- Phe36 stacks with the thymine by wedging through the DNA
- Further recognition of “T” by H-bonding to glutamic acid carboxyl group
- Then, Mismatch binding domains bind mismatch from minor groove aided by Phe-X-Glu binding motif
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Features that ensure MISMATCH recognition
- Intrahelical DNA damage recog. Accounts for fact that the mispaired bases are indistinguishable from normal bases
- Also b/c almost all DNA contacts of MutS are to the DNA phosphate-sugar backbone and sequence
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MutS and Mismatch repair
- Amino (thumb) region is required for MMR
- MutS binds to mismatch or IDL due to higher affinity – binds to phosphate-sugar backbone of DNA
- Binds w/high affinity to G-T and single IDLs (insertion-deletion mismatches): most frequent
- Low affinity for C-C: rarest polymerization error
- MutS may use the DNA duplex destabilization to recognize different mismatches
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MutH and MutL
- Activated by ATP, also through HEMImethylated GATC motif.
- Once activated, now ACTIVATES by CLEAVING UNmethylated DNA strand, leaving 5’ nick on the new DNA strand (unmethylated)
- Same strand is nicked on other side of mismatch
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Three modes of MMR mechanisms
- 1. ATP dependent movement: ATP acts to move MutS-MutL complex along helix
- 2. MutS nucleated polymerization: MutS serves as nucleation site for polymerization of 2nd protein, prob MutL
- 3. DNA bending: MutS-MutL homologues remain bound to mismatch with activation of downstream activities by bending of DNA
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